The goal of our research program is to understand the mechanisms of regulation of biological activity in macromolecular complexes through structural analysis, using X-ray crystallography as our primary tool. (1) Adeno-associated virus (AAV) Rep. Only one animal virus, AAV, integrates its genome into a particular location in human chromosomal DNA. This unique property has important implications for the targeted delivery of genes in the context of gene therapy vectors. Viral integration requires the AAV Rep protein, a multifunctional enzyme that possesses site-specific DNA binding activity, endonuclease activity, ATPase activity, and 3'-to-5' helicase activity. To shed light onto the mechanism of integration, we have previously determined the structures of the endonuclease domain of AAV5 Rep bound to two specific DNA regions found within the three-way DNA junction that marks the end of the viral genome. We are now extending this work to investigate the properties of larger protein-DNA complexes formed by the full-length Rep protein. We have used footprint analysis and protease protection assays to define suitable pieces of DNA on which Rep appears to hexamerize, consistent with its activity as an SF3 helicase; crystals of these complexes have been obtained. (2) DNA transposition. We are interested in the wide variety of solutions that mobile DNA elements have devised to move DNA from one location to another. We are currently investigating several systems including (i) eukaryotic transposons from the hAT (hobo, Ac, Tam3) family; (ii) ISHp608, an insertion sequence originally isolated from Helicobacter pylori; (iii) the ORF1p protein of the human L1 element; and (iv) Sleeping Beauty, a resurrected Tc1/mariner-like element. Hermes, an element isolated from the house fly, has served as a model system for the widely distributed hAT family of transposons, representatives of which are found in fungi, plants, and animals including vertebrates. We recently determined the structure of an active fragment of the Hermes transposase which forms hexamers in solution. We have now defined the smallest portion of transposon-end DNA that can be specifically bound by Hermes, and are currently pursuing the structural characterization of this Hermes-DNA complex. ISHp608 provides an example of another transposition system with unusual features: it transposes using a different mechanism from that of other known transposons as one of the identified reaction intermediates is a circular double-stranded version of the insertion sequence in which the ends are precisely joined to one another. We recently determined the structure of TnpA alone and in complex with a specific DNA sequence that is located close to each end of the insertion sequence. The observed 2:2 complex reveals how the dimeric TnpA is able to bring together both ends of the insertion sequence together, a necessary prelude to moving the DNA segment from one place to another. We are continuing to characterize complexes of TnpA bound to other DNA sequences to provide more snapshots along the chemical reaction pathway. ORF1p is one of two proteins encoded by the mammalian retrotransposon long-interspersed nuclear element (LINE). Although ORF1p is known to bind single-stranded nucleic acids, its precise role in retrotransposition is not yet clear. We hope to provide biochemical insight through structural characterization, and have successfully expressed and purified large quantities of protein suitable for structural work. The same is true of our efforts to understand how the Sleeping Beauty transposase works. This transposase is particularly interesting due to its activity in mammalian cells, a function that has proven useful for insertional mutagenesis studies to locate oncogenes and that has obvious gene therapy applications.